Phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay

Viruses in the genus Coronavirus rw are currently placed in three groups ba sed on antigenic cross-reactivity and sequence analysis of structural prote in genes. Consensus polymerase chain reaction (PCR) primers were used to ob tain cDNA, then cloned and sequenced a highly conserved 922 nucleotide regi on in open reading frame (ORF) Ib of the polymerase (pol) gene from eight c oronaviruses. These sequences were compared with published sequences for th ree additional coronaviruses. In this comparison. it was found that nucleot ide substitution frequencies (per 100 nucleotides) varied from 46.30 to 50. 13 when viruses were compared among the traditional coronavirus groups and, with one exception (the human coronavirus (HCV) 229E), varied from 2.54 to 15.89 when compared within these groups. (The substitution frequency for 2 29E, as compared to other members of the same group, varied from 35.37 to 3 5.72.) Phylogenetic analysis of these pol gene sequences resulted in groupi ngs which correspond closely with the previously described groupings, inclu ding recent data which places the two avian coronaviruses-infectious bronch itis virus (IBV) of chickens and turkey coronavirus (TCV)-in the same group [Guy, J.S., Barnes, H.J., Smith L.G., Breslin, J., 1997. Avian Dis. 41:583 -590]. A single pair of degenerate primers was identified which amplify a 2 51 bp region from coronaviruses of all three groups using the same reaction conditions. This consensus PCR assay for the genus Coronavirus may be usef ul in identifying as yet unknown coronaviruses. (C) 1999 Elsevier Science B .V. All rights reserved.